Recombinant vaccinia viruses that contain varying lengths of dengue DNA coding for the polyprotein NS2B-NS3-NS4A-NS4B-NS5 domain were constructed in order to examine the cleavage strategy that is utilized for expression of this region. Initially, mono-specific antiserum containing antibodies to dengue NS3 or NS5 were prepared by immunization against the appropriate trp E fusion protein. Cells infected with vaccinia recombinant v(NS2B-NS3-NS4A- NS4B-NS5) produced NS2B, NS3, and NS5, whereas vaccinia recombinant v(NS3-NS4A-NS4B-NS5) produced an uncleaved polyprotein encoded by the dengue DNA sequence in the recombinant. Thus, NS2B is necessary for proper processing of the downstream nonstructural proteins. The defect in cleavage of the NS3-NS4A-NS4B-NS5 polyprotein was complemented during coinfection with v(NS2B-30%NS3) or v(NS2B) plus v(30%NS3) as indicated by the production of NS3 and NS5 identifiable by the appropriate antiserum. It appears that cleavage at the NS3-NS4A and NS4B-NS5 junctions of the dengue polyprotein requires functions provided by trans acting NS2B and the N terminal 30% of NS3. Neither NS2B nor NS3 alone is able to mediate these cleavages. Cleavage at the NS4A-NS4B junction appears to be mediated by a specific signalase since a long stretch of hydrophobic sequence precedes the predicted cleavage site. Evidence supporting this cleavage mechanism was also obtained from analysis of the dengue protein products of vaccinia recombinant v(NS4A-NS4B-NS5) infected cells in which cleavage of NS4B-NS5 from the polyprotein was detected. Finally, NS5 antiserum detected a partially cleaved shortened form of NS5 (1-198) in vaccinia recombinant v(NS4B-NS5) or v(NS4B-NS5[1-198]) infected cells. This finding indicates that in the absence of NS2B and NS3, cleavage at the NS4B-NS5 junction can occur at a low level, presumably mediated by an NS4 function. These results suggest that processing of flavivirus nonstructural proteins may utilize more than one cleavage strategy and these cleavage steps are likely complex and highly regulated.